1. Field of the Invention
A novel ligand-mediated immunofunctional assay (LIFA) method is described for detecting the presence and quantitating the amount of a polypeptide hormone binding protein in a biological fluid and/or determining the amount of the ligand polypeptide hormone specifically bound to the hormone binding protein. This modified immunometric assay for a hormone binding protein uses: 1) a first solid phase bound antibody to capture the hormone binding protein; 2) a saturating amount of ligand hormone; and, 3) a labeled second antibody specific for the ligand hormone, The LIFA method is exemplified by a growth hormone binding protein assay.
2. Description of the Background Art
A hormone binding protein (HBP) is a carrier protein found in biological fluids which has binding specificity for a ligand polypeptide hormone. Examples of such HBP's are growth hormone binding protein (GHBP), epidermal growth factor (EGF) binding protein, insulin-like growth factor 1 and 2 (IGF-1, IGF-2) binding proteins (six of them), platelet derived growth factor (PDGF) binding protein, nerve growth factor (NGF) binding protein, insulin binding protein, corticotropin releasing factor (CRF) biding protein, transforming growth factor beta (TGF-.beta.) binding protein and activin binding protein (Follistatin).
One of the best characterized polypeptide hormone binding proteins is the GHBP. The GHBP discussed in this invention is the extracellular domain of the GH receptor which circulates in blood and functions as a GHBP in several species (Ymer, S. I., and Herington, A. C., Mol. Cell. Endocrinol. 41:153 [1985]; Smith, W. C. and Talamantes, F., Endocrinology 123:1489-94 [1988]; Emtner, M., and Roos, P., Acta Endocrinologica [Copenhagen] 122:296-302 [1990], including man (Baumann, G. et. al., J. Clin. Endocrinol. Metab. 62:134-141 [1986]; Herington, A. C. et al., J. Clin. Invest. 77:1817-1823 [1986]). Little is known about the fate of the GHBP or its regulation in various physiological and pathological conditions.
Hormone binding proteins have been assayed by antibody based precipitation methods where the ligand is labeled and the antibody is specific for the binding protein itself. Monoclonal antibodies specific for human growth hormone binding protein (hGHBP) were used by Barnard et al., J. Endocrinol, 123(2):327-32[1989]; for rabbit GHBP by Ymer et al., Endocrinology, 125(2):993-9[1989]; and mouse by Smith et al., Endocrinology 123(3):1489-94[1988]. Currently available methods for estimating GHBP levels in blood are based on incubation of the sample with radiolabeled GH, followed by separation of bound and free GH (Baumann, G. et al., Acta Endocrinologica (Copenhagen) 119: 529-34 [1988]; Amit, T. et al., J. Clin. Endo Metab. 71:474-479 [1990]). The results obtained by these assays are difficult to interpret due to interference by endogenous GH (Baumann, G. et al., J. Clin. Endocrinol. Metab. 62:134-141 [1986]). Others who have used labeled growth hormone to detect GHBP are: Emtner et al., Acta Endocrinol 122(3):296-302, (1990); Silbergeld et al., Clin. Endocrinol. 31(3):295-303 (1989); Daughaday et al., J. Clin. Endocrinol Metab., 65(5):1072-4, (1987); Herington et al., J. Clin. Invest. 77(6):1817-23, (1986); and Laron et al., Acta Endocrinol. 121(4):603-8 (1989). These assays for GHBP in blood have serious problems. They are laborious, requiring separation of complexed GHBP-GH by size-exclusion chromatography or antibody precipitation, and they may not give consistent results from one laboratory to another. In addition, they generate results that are arbitrary (i.e. not calibrated to a common protein standard) and influenced by endogenous growth hormone. Therefore, there is a need for an improved assay method which will allow detection of all the polypeptide hormone binding proteins, including those bound to endogenous polypeptide hormone.
Methods for the production of monoclonal antibody-producing hybridomas are disclosed by Kipps and Herzenberg in Clinical Endocrinology and Metabolism, Vol 62, No. 1, page 108.1-108.9 (1986)
A monoclonal antibody-based immunoradiometric assay for IGF binding protein was described by Pekonen et al, J. Immunoassay 10:325-37 (1989). Immunometric or sandwich immunoassays using high affinity monoclonal antibodies were taught in David et al., U.S. Pat. No. 4,486,530. Such sandwich assays have an antigen with two or more epitopes sandwiched between two antibodies. Circulating proteins that bind non-polypeptide hormone ligands, such as the thyroxine binding protein, have been measured using fluorescent labeled tracer (U.S. Pat. No. 4,476,228) in order to determine the number of binding protein sites not occupied by thyroxine. Iodothyronine immunoassays in a biological fluid using blocking agents and thyroxine binding globulin were described in Gordon et al, U.S. Pat. No. 4,622,293.
Specific binding pairs (SBP) are discussed in reference to antigen-antibody reactions, ligand-receptor, hormone-receptor and lectin-oligosaccharide (U.S. Pat. No. 4,956,302). Zuk et al., (U.S. Pat. No. 4,594,327) describes assays of whole blood to detect members of such SBPs wherein one member of the SBP must be attached to the solid phase prior to contacting the blood. The other second member of the SBP is detected using labeled second SBP member in competitive reactions. Similarly, Weng et al. (U.S. Pat. No. 4,737,456) describes a method of reducing interfering substances in assays of a SBP member wherein the individual member of the SBP is labeled. The receptor is used in a competitive assay to capture both labeled and unlabeled ligand, not to analyze for the presence of and quantify receptor as in the present invention. A method for the determination of an antigen using two antibodies is disclosed in U.S. Pat. No. 4,343,896.
Problems in Previous Polypeptide Hormone Binding Protein Assays
Problems in detecting HBP can be best illustrated by the problems encountered in detecting GHBP. Previous methods for determining the presence of GHBP in biological fluids were not as accurate as desired and frequently required the use of radioactive materials. The present invention avoids the problems of the previous assay methods in that it (a) does not require the use of radiolabeled GH; (b) does not require the removal of endogenous GH from the GHBP; (c) does not require any form of size separation of GH from the GH-GHBP complex; and, (d) measures the actual mass or absolute amount of GHBP rather than a relative amount reported in arbitrary units. The present invention has the added advantage that it measures the binding capacity of the circulating GHBP and is able to measure the degree of saturation of the GHBP with respect to GH. Moreover, the present invention is specific for GHBP by substantially reducing background assay noise that causes imprecision. The assay of the present invention avoids the problems in standard immuno-metric assays by using a first antibody to capture the GHBP and a second detectably labeled antibody to measure the present of bound GH. The use of a second antibody specific for another epitope on the GHBP would not determine whether the GHBP was functional, and in addition, it could increase the background due to other serum proteins which bound both antibodies.
In order to study the function of the endocrine system it is essential to have access to reliable methods for quantitation of all parts of the system, i.e. the hormone, its being protein and the hormone-binding protein complex. These measurements have not been previously achieved because of interference in the assays by the different components and the fact that both the hormone and the BP can be present in free and complexed forms. It is even more complicated when there are several different binding proteins for the same hormone, as for the IGF-1 system. However, the present invention teaches how the use of monoclonal antibodies directed at a specific binding protein can measure the amount and degree of saturation of that specific binding protein. In the case of GH, for which a second binding protein with lower affinity has been described (Baumann, G. and Shaw, M. A., J. Clin. Endocrinol. Metab. 70:680-686 [1990]), this binding protein appears to be structurally unrelated to the GH receptor and should not be detected in our assay. However, this other GH binding protein may also be detected in the present invention's assay method once the other binding protein is isolated and appropriate antibodies are raised.
Currently, the standard method for quantitation of carrier proteins for peptide hormones is incubation of serum with the radiolabeled ligand, followed by chromatography or precipitation to separate the bound and free hormone. These procedures are laborious and the results often difficult to interpret because of the interference by endogenous hormone in the sample. These assay methods give an estimate of the binding capacity of serum proteins of a certain size but the activities of different binding proteins of similar size are not distinguished and the relative proportion of free and complexed BP cannot be determined. Another disadvantage is that the results are expressed in relation to reference serum pools, which makes it difficult to compare the results in different studies. In the present invention, we developed an assay for the GHBP by choosing a new approach. This assay was surprisingly able to precisely detect individual hormone binding proteins in a way that has not been previously demonstrated.
The LIFA, which is the preferred method used in the present invention, is simple to use and has the advantage that only functional binding protein is detected. When the assay method is applied to GHBP, both total and endogenously complexed GHBP are measured, and the assay does not require removal of endogenous GH from the GHBP or procedures to separate free GH from the GHBP complex. In contrast to previous methods, endogenous GH does not interfere in the assay; instead, bound GH, either endogenous or exogenously added, is used to detect the total and complexed GHBP. In fact, one cannot use the GH as the first member bound to the solid phase since GH cannot complex with binding protein that is already complexed with endogenous GH. Therefore, one requirement of the present assay method is for a solid phase coat antibody which recognizes the binding protein both in a free and complexed form. The assay method taught in this invention can also be used to measure the total binding capacity and the saturation of other polypeptide hormone-binding proteins with respect to the ligands that they bind.
Therefore, the present invention describes the development of a novel, sensitive and specific enzyme-linked immunosorbent assay (ELISA) for quantitation of biologically active HBP in biological fluids. The assay can also be used to measure the concentration of the ligand-hormone binding protein complex. The method of the present invention provides a number of advantages relating to ease of analysis, sensitivity, precision and reliability which will be more apparent as the details of the method are discussed.